LPS is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. LPS is a potent inducer of inflammation, stimulating the expression of many pro-inflammatory and pro-coagulant mediators in monocytes, macrophages and endothelial cells. These responses are important in containing and eliminating a localized infection, however, adverse effects of systemic exposure to LPS can include induction of an inflammatory cascade, damage to endothelium, widespread coagulopathies, and organ damage. Systemic exposure to LPS can arise from direct infection of gram negative bacteria, leading to the complications of gram-negative sepsis [Tracey et al., Adv. Surg. 23: 21-56 (1990)]. Alternatively, a variety of conditions and circumstances, including trauma, can induce changes in gut permeability that result in translocation of bacteria, and therefore LPS, into circulating blood. Bacterial LPS translocated from the gut is thought to play a major role in post-surgical immunosuppression [Little et al., Surgery 114(1): 87-91 (1993)] and hemorrhagic shock. Therefore, there exists a need to discover therapies that can counteract the effects of LPS in pathologic situations.
Two proteins, CD14 and lipopolysaccharide binding protein (LBP) [Schumann et al., Science 249: 1429-1431 (1990); Wright et al., Science 249: 1431-1433 (1990)] have been shown to be required to generate an inflammatory response to LPS. LPS must bind to CD14 to activate an inflammatory response. CD14 is a 55 kD protein expressed via a glycosylphosphatidylinositol-anchor on the surface of macrophages, monocytes and neutrophils (mCD14). Endothelial and epithelial cells, which do not express the CD14 protein, are activated by LPS bound to a soluble form of (sCD14) found in serum or plasma (at a concentration of about 2 .mu.g/mL in normal human blood). CD14 preferentially binds to LPS monomers [Tobias et al., J. Biol. Chem. 270(18): 10482-10488 (1995)]. Since purified LPS exists in aqueous solution in micelles or aggregates, direct binding of LPS to CD14 is very slow [Tobias et al. (1995), supra; Yu and Wright, J. Biol. Chem. 271(8): 4110-4105 (1996)] and only occurs at high concentrations of LPS [Hailman et al., J. Exp. Med. 179(1): 269-277 (1994)]. Binding of LPS to CD14 is greatly accelerated by LBP [Hailman et al. (1994), supra; Tobias et al. (1995), supra; Yu et al. (1996), supra], and LBP is required for activation of cells by either mCD14 or sCD14 at physiological concentrations of LPS [Schumann et al. (1990), supra; Wright et al. (1990), supra].
LBP is a 60 kD glycoprotein synthesized in the liver and present in normal human serum. LBP belongs to the group of plasma proteins called acute phase proteins, including C-reactive protein, fibrinogen and serum amyloid A, that increase in concentration in response to infectious, inflammatory and toxic mediators. LBP expression has been induced in animals by challenge with LPS, silver nitrate, turpentine and Corynebacterium parvum [Geller et al., Arch. Surg. 128(1): 22-28 (1993); Gallay et al., Infect. Immun. 61(2): 378-383 (1993); Tobias et al., J. Exp. Med. 164: 777-793 (1986)]. However, while administration of silver nitrate caused LBP levels to increase in several strains of mice, this was not observed in one strain, C3H/HeJ, in which LPS does not induce an inflammatory response [Gallay et al. (1993), supra]. Recently, an analysis of different human disease states has indicated that increased LBP levels are uniquely correlated with exposure to LPS. In human patients with presumed gram-negative sepsis, serum LBP levels can reach from about 50 to about 100 .mu.g/mL [U.S. Pat. No. 5,484,705]. In contrast, in other disease states, such as rheumatoid arthritis, involving an acute phase response in which elevated levels of the acute phase proteins CRP and fibrinogen were measured in patient serum samples, no significant increases in LBP levels were observed. Elevated, particularly persistently elevated, LBP levels have been correlated with poor clinical outcome in septic patients [U.S. Pat. No. 5,484,705, and U.S. Ser. No. 08/377,391 filed Jan. 24, 1995, now U.S. Pat. No. 5,804,367, both of which are hereby incorporated by reference in their entirety. This has been confirmed by Schumann et al., 36th Int'l Conf. on Antimicrobial Agents and Chemotherapy, New Orleans, La., Sep. 15-18, 1996.
LBP is reported to bind to LPS aggregates (at low LBP to LPS ratios) or to disaggregate LPS vesicles (at high LBP to LPS ratios) [Tobias et al. (1995), supra] to form an LBP:LPS complex that greatly facilitates binding of LPS to either mCD14 or sCD14 [Wright et al., J. Exp. Med. 173(5): 1281-1286 (1991); Hailman et al. (1994), supra; Yu et al. (1996), supra; Tobias et al. (1995), supra]. LBP is reported to act catalytically in facilitating LPS binding to CD14, a single LBP molecule enabling the transfer over 100 LPS molecules to CD14 [Hailman et al. (1994), supra]. LBP is also reported to remain associated with LPS aggregates or LPS coated particles and facilitate binding to cells expressing mCD14 in a phenomenon known as opsonization [Wright et al., J. Exp. Med. 170(4): 1231-1241 (1989); Kirkland et al., J. Biol. Chem. 268(33): 24818-24823 (1993); Gegner et al., J. Biol. Chem. 270(10): 5320-5325 (1995)]. Thus, LBP potentiates the inflammatory activity of LPS and is recognized as an immunostimulatory molecule. Functional analysis of the LBP molecule has demonstrated that LPS binding resides in the approximate N-terminal half of the protein, but the C-terminal half is required to permit transfer of LPS to CD14 [U.S. application Ser. No. 08/261,660 filed Jun. 17, 1994 now U.S. Pat. No. 5,731,415, Theofan et al., J. Immunol. 152(7): 3624-3629 (1994); Han et al., J. Biol. Chem. 269(11): 8172-8175 (1994)]. Because of the observed potentiating effect LBP has on the inflammatory potential of LPS, blocking or interfering with the immunostimulatory activity of LBP has been a therapeutic target of interest.
For example, a polyclonal antibody preparation to murine LBP has been shown to prevent LBP mediated binding of LPS to murine macrophages and subsequent induction of TNF expression in vitro, effectively neutralizing the activity of LBP. This same polyclonal antibody was able to reduce lethality in a murine model of endotoxemia [Gallay et al. (1993), supra].
Several modified forms of LBP have been developed that bind LPS but lack the ability to transfer the LPS molecule to CD14. U.S. application Ser. No. 08/261,660 filed Jun. 17, 1994, now U.S. Pat. No. 5,731,415, hereby incorporated by reference in its entirety, describes novel biologically active polypeptide derivatives of LBP, including LBP derivative hybrid proteins, which are characterized by the ability to bind to LPS and which lack CD14-mediated immunostimulatory properties, including the ability of LBP holoprotein to mediate LPS activity via the CD14 receptor. More particularly, these LBP protein derivatives including LBP derivative hybrid proteins lacking those carboxy terminal-associated elements characteristic of the LBP holoprotein which enable LBP to bind to and interact with the CD14 receptor on monocytes and macrophages so as to provide an immunostimulatory signal to monocytes and macrophages. Such LBP protein derivatives included those characterized by a molecular weight less than or equal to about 25 kD, including an amino-terminal LBP fragment having amino acid residues 1-197 that was designated rLBP.sub.25. This recombinant protein corresponding to the amino-terminal residues 1-197 of LBP has been shown to bind LPS but could neither facilitate binding of LPS to CD14 nor permit LPS-induced expression of TNF [see also, Theofan et al. (1994), supra; Han et al. (1994), supra]. Additionally, this N-terminal fragment was shown to inhibit LPS-induced expression of TNF that was mediated by full-length LBP [Han et al. (1994), supra]. rLBP.sub.25 includes amino acid regions comprising LBP residues 17 through 45, 65 through 99 and 141 through 167 which correspond to respective biologically active (e.g., LPS binding) domains (e.g., Domain I--residues 17 through 45; Domain II--residues 65 through 99; and Domain III--residues 142 through 169) of bactericida/permeability-increasing protein (BPI). The LBP derivative hybrid proteins included hybrids of LBP protein sequences with the amino acid sequences of other polypeptides and also characterized by the ability to bind to LPS and the absence of CD14-mediated immunostimulatory properties. Such hybrid proteins included fusions of LBP amino-terminal fragments with polypeptide sequences of other proteins such as BPI, immunoglobulins and the like. Properties of several LBP/BPI fusion proteins have been described by Abrahamson et al., J. Biol. Chem. 272(4):2149-2155 (1997). In addition, a recombinant hybrid fusion between the N-terminal 199 amino acid residues of LBP and the C-terminal 257 residues of BPI was shown to be protective in a rodent model of gram-negative sepsis [Opal et al., Antimicrob. Agents Chemother. 39(12): 2813-2815 (1995)]. U.S. application Ser. No. 08/261,660 filed Jun. 17, 1994 also describes LBP derivatives in the form of synthetic LBP peptides that are portions of the LBP sequence corresponding to either Domain II (residues 65-99) or Domain III (residues 142-169) of BPI. The LBP derivative designated LBP-1 consisted of residues 73 through 99 of LBP. The LBP derivative designated LBP-2 consisted of residues 140 through 161 of LBP. In addition, Taylor et al., J. Biol. Chem. 270(30): 17934-17938 (1995), described synthetic peptides corresponding to residues 91-105 or 94-108 of the mature LBP protein that were reported to compete with LBP for binding to LPS and could inhibit LPS-induced expression of TNF in vitro.
In addition to transferring LPS to CD14, LBP can facilitate the transfer of LPS to serum lipoproteins [Wurfel et al., J. Exp. Med. 180: 1025-1035 (1994)]. Association with lipoproteins greatly reduces the inflammatory potential of LPS [Ulevitch and Johnston (1978), supra]. Thus, LBP itself can also participate in the neutralization of LPS. The significance of LBP-mediated transfer of LPS to lipoproteins, however, remains unclear. Specifically, elevated levels of LBP found in acute phase serum have been correlated with a reduction of the rate of association of LPS with lipoproteins [Tobias and Ulevitch, J. Immunol. 131(4): 1913-1916 (1983); Tobias et al. (1985), supra; U.S. Pat. Nos. 5,245,013 and 5,310,879]. This ability of LBP to inhibit, rather than facilitate, the transfer of LPS to lipoproteins was exploited in the initial purification of LBP [Tobias et al. (1986), supra].
Dedrick et al., J. Endotoxin Research 3(supp. 1):18 (Abstract I-14) (October 1996) reported in an abstract that concentrations of 1 ng/mL to 1 .mu.g/mL of rLBP fully potentiated the induction of TNF expression in serum-free medium by 1 ng/mL LPS on a human monocytic cell line (THP.1). In medium containing 10% serum, LBP concentrations of 30 .mu.g/mL or greater inhibited LPS-induced TNF expression by the THP.1 cells and also inhibited E-selection expression in human umbilical endothelial cells (HUVEC) induced by 10 ng/mL LPS. Moreover, it was reported that administration of 5 mg/kg rLBP also increased survival in mice challenged with up to 25 mg/kg E. coli LPS. However, human subjects suffering from disorders involving bacteria and their endotoxin (such as sepsis) have been shown to exhibit substantially elevated levels of LBP in circulation (at concentrations of 50 .mu.g/mL to 100 .mu.g/mL of serum), yet these high circulating levels of LBP do not appear to have inhibited the adverse effects of bacterial endotoxin in circulation that were experienced by these subjects. The role of LBP in promoting or alleviating adverse effects of endotoxin in circulation thus remains unclear.
Bactericidal/permeability-increasing protein (BPI) is a basic protein found in the azurophilic granules of polymorphonuclear leukocytes [Weiss et al., J. Biol. Chem. 253(8): 2664-2672 (1978)]. BPI binds to LPS, resulting in its clearance and neutralization. The amino acid sequence of BPI is closely related to that of LBP [Schumann et al. (1990), supra], and like LBP, the amino-terminal half of BPI has a binding site for LPS [Ooi et al., J. Exp. Med. 174: 649-655 (1991)]. However, BPI has a higher affinity for LPS than does LBP [Gazzano-Santoro et al., Infect. Immun. 62(4): 1185-1191 (1994); Wilde et al., J. Biol. Chem. 269(26): 17411-17416 (1994)], and cannot transfer LPS to the CD14 molecule. Thus, BPI effectively competes with LBP for LPS binding [Heumann et al., J. Infect. Dis. 167: 1351-1357 (1993); Gazzano-Santoro et al. (1994), supra] and blocks the inflammatory activity of LPS in vitro [Marra et al., J. Immunol. 144(2): 662-666 (1990); Ooi et al. (1991), supra], and in humans [de Winter et al., J. Inflamm. 45: 193-206 (1995)].
It has been suggested that sCD14 could be a useful therapeutic agent in endotoxin-related disorders [Schutt et al. (1991), supra; Schutt et al. (1992), supra; Haziot et al. (1994), supra; Haziot et al. (1995), supra]. The presence of sCD14 reduces the amount of LPS complexed with LBP [Tobias et al. (1995), supra], because, although LBP has a higher affinity for LPS than sCD14, the distribution of LPS between sCD14 and LBP depends on the molar ratio of the two proteins. sCD14 has been shown to inhibit responses that depend on mCD14 [Schutt et al., Res. Immunol. 143: 71-78 (1992); Schutt et al., Allerg. Immunol. 37: 159-164 (1991); Haziot et al., J. Immunol. 152: 5869-5876 (1994)], and to protect mice against experimental endotoxemia [Haziot, et al., J. Immunol. 154: 6529-6532 (1995)].